A Large Survey of Croatian Wild Mammals for Giardia duodenalis Reveals a Low Prevalence and Limited Zoonotic Potential

Abstract

Wild mammals are considered an important source of potentially zoonotic Giardia duodenalis parasites, yet surprisingly little information is available on the actual prevalence and the genetic identity of the species they harbor. A large survey was conducted in Croatia by collecting 832 fecal samples from red deer (Cervus elaphus, n = 374), roe deer (Capreolus capreolus, n = 21), wild boars (Sus scrofa, n = 144), foxes (Vulpes vulpes, n = 66), bears (Ursus arctos, n = 19), wolves (Canis lupus, n = 127), jackals (Canis aureus, n = 8), and hares (Lepus europeus, n = 73). Fecal samples were tested for the presence of Giardia cysts using fluorescent microscopy. The observed prevalence ranged from low (1% in red deer, 1.7% in wild boars, and 4.5% in foxes) to moderate (10% in wolves and 12.5% in jackals) to high (24% in roe deer). No cysts were observed in bears and hares. Polymerase chain reaction was performed on microscopically positive samples to amplify fragments of the small subunit ribosomal gene, the ribosomal 5.8S gene and the two flanking internal transcribed sequences, and the triose phosphate isomerase gene. Sequence analysis showed a predominance of G. duodenalis assemblage A in both ruminants (genotypes A1 and A3) and carnivores (genotype A1). G. duodenalis assemblages B, C, and D, as well as Giardia microti, were also detected in this study. This is the first molecular description of the parasite from the red deer, the wolf, and the jackal. The data point to a minor role of wild mammals as reservoirs of zoonotic assemblages of G. duodenalis, albeit cycling between sylvatic and domestic animals is possible.

Introduction

G iardiasis is a common infection of vertebrates including humans, and its complex epidemiology is only partially understood (Cacciò and Ryan 2008). In mammals, three species of the genus Giardia are recognized, two of which, Giardia microti and Giardia muris, parasitize various rodent species, whereas the third, Giardia duodenalis, has a broad host range that includes humans, domestic animals, and wildlife (Thompson and Monis 2004).

It is now well established that the morphologically invariant G. duodenalis species comprises at least seven major genetic groups, referred to as assemblages A to G, which may represent cryptic species (Monis et al. 2009). Among these, assemblages A and B are found in both humans and animals, whereas assemblages C and D are found in dogs and other carnivores, assemblage E in livestock, assemblage F in cats, and assemblage G in rats (Monis et al. 2009). Recently, the presence of another assemblage (assemblage H) in marine mammals has been suggested (Lasek-Nesselquist et al. 2010).

Understanding the role played by animals in the epidemiology of human infection is an important yet unresolved issue (Sprong et al. 2009). Despite the fact that the WHO has recognized G. duodenalis as a potentially zoonotic agent since 1979 (WHO Expert Committee 1981), and the suggested role of aquatic mammals in the waterborne transmission to humans (Wallis et al. 1984), little information is available on the Giardia species and G. duodenalis assemblages that infect wildlife (Olson and Buret 2001). Some recent studies, which have been supported by molecular analyses (Sulaiman et al. 2003, Fayer et al. 2006, Hamnes et al. 2007, Lalle et al. 2007, Robertson et al. 2007, Lasek-Nesselquist et al. 2008, Lebbad et al. 2010), have demonstrated the presence of both host-adapted and zoonotic G. duodenalis genotypes in different species of wild animals, but the picture is still largely fragmentary.

In the present work, a large number of fecal samples have been collected in Croatia from wild carnivores (wolves, foxes, bears, and jackals), wild hoofed animals (wild boars, red deer, and roe deer), and wild hares, tested for the presence of Giardia cysts, and further characterized by molecular methods.

Materials and Methods

Source of isolates

Fecal samples were collected in Croatia from red deer (Cervus elaphus, n = 374), roe deer (Capreolus capreolus, n = 21), wild boars (Sus scrofa, n = 144), foxes (Vulpes vulpes, n = 66), bears (Ursus arctos, n = 19), wolves (Canis lupus, n = 127), jackals (Canis aureus, n = 8), and hares (Lepus europeus, n = 73). The approximate locations of the sampling areas are shown in Figure 1. Samples from red deer, wild boars, and roe deer were collected from 2006 to 2009, samples from foxes from November 2007 to February 208, samples from bears in 2007, samples from wolves from 2005 to 2009, samples from jackals in 2007 and 2008, and samples from hares in 2006. Most of the samples were collected from the rectum of shot animals, or after necropsy, during the hunting season in the periods reported above. Animals were shot according to the management plan approved by the Governmental Institution for the management of game species. For 80 (21.4%) of the 374 red deer samples and 67 (52.7%) of the 127 wolf isolates, fecal samples were collected from the ground at 10 different locations, to avoid multiple sampling of the same individual. The age of animals was as follows: for red deer from 8 months up to 9 years, for roe deer from 7 months up to 5 years, for wild boars from 5 months up to 4 years, for hares from 6 months up to 2 years, for bears over 3 years, for foxes from 1 to 4 years, for jackals from 1 to 5 years, and for wolves from 5 months up to 4 years.

FIG. 1.

Map of Croatia showing the approximate locations of the sampling areas for the different host species.

Cyst isolation, microscopy, and DNA isolation

Fecal samples were concentrated by flotation on a sucrose gradient (specific gravity, 1.06), and the presence of Giardia spp. cysts was assessed by an immunofluorescence (IF) assay using a commercial kit according to the manufacturer's instructions (Merifluor; Meridian Bioscience, Cincinnati, OH). Only one fecal sample per animal was examined. DNA was extracted from fecal samples after the flotation step according to a previously described procedure (da Silva et al. 1999). Briefly, samples were homogenized using the FP120 Fast Prep Cell disruptor (Savant; Thermo Electro Corporation, Woburn, MA). The DNA released after the lysis step was purified using the Fast DNA extraction kit (Qbiogene, Illkirch Cedex, France) and eluted in a final volume of 50 μL.

Molecular methods

A nested polymerase chain reaction (PCR) assay was used to amplify a fragment of the small subunit ribosomal RNA (ssu-rDNA) following the procedure described by Read et al. (2002). A recently developed nested PCR assay that amplifies the region encompassing the 5.8 gene and the flanking internal transcribed sequences 1 and 2 (ITS1 and ITS2) was also used (Cacciò et al. 2010). For the amplification of a fragment of the triose phosphate isomerase (tpi) gene, the protocol described by Sulaiman et al. (2003) and the assemblage D-specific protocol described by Lebbad et al. (2010) were used. In all cases, the primary PCR reaction consisted of 25 μL of 2 × PCR master mix (Promega, Milan, Italy), 10 pmol of each primer, and 1–3 μL of DNA in a total reaction volume of 50 μL. For the nested PCR, 2.5–5 μL of the first PCR was used as template. Positive (DNA from trophozites of cultured WB and GS strains, representing assemblages A and B, respectively) and negative (water) controls were included in each experiment. All PCR assays were repeated at least two times (three times in the case of tpi).

PCR products were separated by electrophoresis in 1.5% agarose gels stained with ethidium bromide, purified using spin columns, and sequenced on both strands. Sequences were assembled using the SeqMan 7.0 program in the Lasergene software package (DNASTAR, Madison, WI). Novel sequences have been deposited in the GenBank database under the accession numbers HQ259661, HQ259662, and HQ259663.

Results

Prevalence of Giardia spp. in wild mammals

Table 1 displays the prevalence of Giardia cysts as found in the feces of the 832 mammals tested by IF microscopy. The observed prevalence ranged from low (1.7% in wild boars and 4.5% in foxes) to moderate (10% in wolves and 12.5% in jackals) to high (24% in roe deer). Samples from bears and hares were negative. The number of cysts in all samples, as judged qualitatively by microscopy, was generally low (less than five cysts per microscopic field).

Table 1.

Prevalence of Giardia spp. in Fecal Samples Collected from Wild Mammals in Croatia

Host	No. of samples/no. of positive	Prevalence (95% confidence intervals)
Red deer	374/4	1.1 (0.4%–3.1%)
Roe deer	21/5	24 (8.2%–47%)
Wild boar	144/2	1.4 (0.2%–4.9%)
Fox	66/3	4.6 (1.0%–12.7%)
Bear	19/0	0 (0%–17.6%)
Wolf	127/13	10.2 (5.6%–16.9%)
Jackal	8/1	12.5 (0.3%–53%)
Hare	73/0	0 (0%–4.9%)
Total	832/28	3.3 (2.4%–5.0%)

Values within parentheses indicate 95% exact binomial confidence intervals.

Molecular characterization at the SSU-rRNA locus

DNA was extracted from the 26 microscopically positive samples and submitted to amplification of a SSU-rRNA gene fragment for identification of Giardia species. A total of 23 isolates (88.5%) yielded a fragment of the expected size. Sequence analysis revealed the presence of G. microti in a wolf and a roe deer, G. duodenalis assemblage A in 14 isolates (3 from red deer, 2 from roe deer, 1 from a wild boar, 6 from wolves, 1 from a fox, and 1 from a jackal), G. duodenalis assemblage C in 2 wolves, and G. duodenalis assemblage D in 2 roe deer, a red deer, and a wolf (Table 2). A mixed A+C infection was detected, on the basis of overlapping peaks at positions 63 (T in assemblage A, A in assemblage C) and 73 (C in assemblage A, G in assemblage C), in a wolf (Table 2).

Table 2.

Genotyping Results at Three Loci of Giardia spp. from Wild Mammal Isolates

Host	Code	SSU-rDNA	ITS1-5.8S-ITS2	TPI/TPI (D)
Red deer	ISSGDA820	Neg	A1	Neg/neg
Red deer	ISSGDA821	A	A1	A
Red deer	ISSGDA867	A	Neg	Neg/neg
Red deer	ISSGDA868	A	A3	Neg/neg
Red deer	ISSGDA829	D	Neg	Neg/neg
Roe deer	ISSGDA182	Giardia microti	Neg	C
Roe deer	ISSGDA815	D	Neg	Neg/neg
Roe deer	ISSGDA816	A	Neg	A1
Roe deer	ISSGDA830	D	Neg	Neg/neg
Roe deer	ISSGDA869	A	A3	Neg/neg
Wild boar	ISSGDA688	A	A3	A3
Wolf	ISSGDA702	A	A1	B/D
Wolf	ISSGDA703	C	C	C
Wolf	ISSGDA704	A+C	Avar	Neg/D
Wolf	ISSGDA705	A	A1	A1
Wolf	ISSGDA706	A	A1	Neg/neg
Wolf	ISSGDA707	A	A1	A1
Wolf	ISSGDA822	G. microti	Neg	Neg/neg
Wolf	ISSGDA823	D	C	Neg/D
Wolf	ISSGDA824	Neg	A1	Neg/neg
Wolf	ISSGDA825	Neg	D	Neg/neg
Wolf	ISSGDA826	A	A1	Neg/neg
Wolf	ISSGDA827	A	Neg	Neg/neg
Wolf	ISSGDA828	C	C	Neg/neg
Fox	ISSGDA837	A	Neg	Neg/neg
Jackal	ISSGDA831	A	Neg	B

For the tpi gene, data obtained using either generic or assemblage D-specific primers are shown.

ITS, internal transcribed sequences.

Molecular characterization at the 5.8S and ITS locus

The same samples were submitted to amplification of the fragment encompassing the 5.8S gene and the two flanking ITS1 and ITS2. A total of 16 isolates (61.5%) gave a clear amplification product. Sequencing revealed assemblage A in 12 isolates: genotype A1 (from subassemblage AI) was detected in 8 isolates (from 2 red deer and 6 wolves); genotype A3 (from subassemblage AIII) in 3 isolates (from a red deer, a roe deer, and a wild boar); and a novel variant within assemblage A, characterized by a 4 bp deletion in the ITS2 region, was found in a wolf isolate (ISSGdA704). This latter variant was confirmed by sequencing three independent amplification products (GenBank accession number: HQ259663). Assemblage C was detected in three wolves, and assemblage D in a single wolf isolate (Table 2).

Molecular characterization at the tpi locus

Amplification of a tpi gene fragment was successful in nine isolates (34.6%) using generic primers, whereas three samples (11.5%) from carnivores were amplified only when using the assemblage D-specific primers (Table 2). Assemblage A was found in five isolates: genotype A1 was detected in a roe deer and two wolves, genotype A3 in a wild boar, and a novel genotype (differing from A1 by four single-nucleotide polymorphisms [SNPs]) was detected in a red deer (isolate ISSGdA821; GenBank accession number: HQ259661).

Assemblage B was found in a wolf and a jackal: the sequence from the wolf was identical to genotype BIV, whereas the sequence from the jackal showed four SNPs compared with genotype BIV and represents a novel variant (isolate ISSGdA831; GenBank accession number: HQ259662). Assemblage C was found in a roe deer and a wolf: the sequences showed two and three SNPs, respectively, compared with a sequence from a dog, which represent the closest match found in the GenBank database (AY228641). Finally, assemblage D was found in three wolves (Table 2). Of note, one of the three wolf isolates was typed as assemblage B by PCR and sequencing using generic primers (Table 2).

Discussion

As stated in the most recent review on Giardia in wildlife (Appelbee et al. 2005), “one of the prominent questions driving research in wildlife parasitology is whether or not a wildlife population can serve as a reservoir of disease for humans and domestic animals.” This question can only be addressed by combining prevalence data with a molecular characterization of the isolates and by conducting studies in well-defined locations where the animal/environment interactions are known (Thompson et al. 2010). To date, few studies have used PCR and sequence analysis to identify the Giardia species or G. duodenalis assemblages present in fecal samples of wild animals. The results have demonstrated the occurrence of the G. duodenalis zoonotic assemblages A and B in marine (dolphin, porpoise, and seal), aquatic (beaver and muskrat), and terrestrial (several species of carnivores, wild hoofed animals, and marsupials) mammals, whereas the host-specific assemblages C and D have been detected in coyotes, and G. microti in rodents (a summary of the literature data is presented in Table 3).

Table 3.

Literature Data on the Prevalence and Molecular Characterization of Giardia spp. in Wild Mammals

Host (common name)	Origin	No. of samples	Prevalence	Giardia species/Giardia duodenalis assemblage	Gene(s) studied	Reference
Castor canadensis (beaver)	United States	62	1.5%	Assemblage B	18S rRNA, bg, tpi	Fayer et al. (2006)
	United States	7	n.a.	Assemblage B	18S rRNA, tpi	Sulaiman et al. (2003)
Ondatra zibethicus (muskrat)	United States	8	n.a.	G. microti, Assemblage B	18S rRNA, tpi	Sulaiman et al. (2003)
Rattus norvegiscus (brown rat)	Sweden	10	n.a.	Assemblage G, G. muris	18S rRNA, bg	Lebbad et al. (2010)
Mus musculus (common mouse)	Sweden	2	n.a.	G. muris, G. microti	18S rRNA	Lebbad et al. (2010)
Dama dama (fallow deer)	Italy	139	11.5%	Assemblage A	bg, tpi	Lalle et al. (2007)
	Sweden	1	n.a.	Assemblage A	bg, tpi, gdh	Lebbad et al. (2010)
Alces alces (wild moose)	Norway	455	12.3%	Assemblage A	bg, gdh	Robertson et al. (2007)
Rangifer tarandus (reindeer)	Norway	188	7.3%	Assemblage A	bg, gdh	Robertson et al. (2007)
Ovibos muschatus (muskox)	Canada	72	21%	Assemblage A	18S rRNA	Kutz et al. (2008)
Odocoileus virginiasus (white-tailed deer)	United States	26	3.8%	Assemblage A	bg, tpi	Trout et al. (2003)
Bos grunniens (yak)	Sweden	1	n.a.	Assemblage E	bg, tpi, gdh	Lebbad et al. (2010)
Vulpes vulpes (red fox)	Norway	269	4.8%	Assemblages A, B	bg, gdh	Hamnes et al. (2007)
	Australia	1	n.a.	Assemblage A	18S rRNA	McCarthy et al. (2008)
Canis latrans (coyote)	United States	22	32%	Assemblages B, C, D	18S rRNA	Trout et al. (2006)
	Canada	70	18.6%	Assemblage A, D	18S rRNA, gdh	Thompson et al. (2009)
Alouatta pigra (black howler monkey)	Mexico	66	31%	Assemblages A, B	18S rRNA	Vitazkova and Wade (2006)
Gorilla gorilla beringei (mountain gorilla)	Uganda	100	2%	Assemblage A	18S rRNA	Graczyk et al. (2002)
Macaca fuscata (Japanese macaque)	Japan	3	n.a.	Assemblage B	gdh	Itagaki et al. (2005)
Procolobus badius tephrosceles (red colobus)	Uganda	30	11%	Assemblages B. E	18S rRNA, ef-1α, tpi, gdh	Johnston et al. (2010)
Colobus guereza (black and white colobus)	Uganda	29		Assemblage B
Cercopithecus ascanius (red-tailed guenon)	Uganda	22		Assemblage B
Macropus fuliginosus (western gray kangaroo)	Australia	72	4.2%	Assemblage A	18S rRNA	McCarthy et al. (2008)
	Australia	105	4.8%	Assemblages A, B	18S rRNA, gdh	Thompson et al. (2008)
Isoodon obesulus (quenda)	Australia	72	1.5%	Novel genotype	18S rRNA, ef-1α	Adams et al. (2004)
Trichosurus cunninghami (mountain brushtail)	Australia	76	25%	Assemblage A	18S rRNA, gdh	Thompson et al. (2008)
T. vulpecula (silver gray brushtail possum)	Australia
Phascolarctos cinereus (koala)	Australia	11	27.3%	Assemblage A	18S rRNA, gdh	Thompson et al. (2008)
Wallabia bicolor (swamp wallaby)	Australia	16	12.5%	Assemblage A	18S rRNA, gdh	Thompson et al. (2008)
Phoca groenlandica (harp seal)	United States	1	n.a.	Assemblages A, B	bg, tpi, gdh, mlh1	Lasek-Nesselquist et al. (2008)
	Canada	38	42%	Assemblage A	18S rRNA	Appelbee et al. (2010)
Phoca vitulina (harbor seal)	United States	1	n.a.	Assemblage B	bg, tpi, gdh, mlh1	Lasek-Nesselquist et al. (2008)
Delphinus delphis (common dolphin)	United States	4	n.a.	Assemblages A, B	bg, tpi, gdh, mlh1	Lasek-Nesselquist et al. (2008)
Lagenorhynchus acutus (white-sided dolphin)	United States	3	n.a.	Assemblage B	bg, tpi, gdh, mlh1	Lasek-Nesselquist et al. (2008)
Grampus grisou (Risso's dolphin)	United States	1	n.a.	Assemblage B	bg, tpi, gdh, mlh1	Lasek-Nesselquist et al. (2008)
Phocoena phocoena (harbor porpoise)	United States	3	n.a.	Assemblages A, B	bg, tpi, gdh, mlh1	Lasek-Nesselquist et al. (2008)

n.a., not assessed.

In the present work, the prevalence of Giardia spp. cysts was estimated using IF microscopy on 832 fecal samples collected from different wild mammals of Croatia (Fig. 1). Our data, and those from previous studies, indicate a moderately high prevalence (7%–27%) in certain wild hoofed animals (roe deer, fallow deer, moose, and reindeer) but not in others (red deer, white-tailed deer, and wild boars). Among carnivores, the prevalence in foxes from Norway (Hamnes et al. 2007) and Croatia (present study) were similar and low (4.8% and 4.5%, respectively), whereas a higher prevalence was estimated in wolves (10%) and jackals (12.5%) in Croatia and even higher (32%) in coyotes from the United States (Trout et al. 2006). Most of the animals tested in this study were adult (>6 months of age), as hunting young animals is forbidden in Croatia and hunters often hunt adult animals for trophy. As the prevalence and intensity of infection are higher in young compared with adult hosts (Olson and Buret 2001), the low prevalence observed in this study, and the low number of cysts present in fecal samples, may in part be attributed to this sampling effect. However, in carnivores, this could also reflect exposure of the host to different species/assemblages of Giardia as a result of differences in prey choice; for example, animals that prey on rodents (such as foxes) are more likely to ingest cysts of G. muris, G. microti, or G. duodenalis assemblage G, which may be unable to establish infection in these hosts, therefore accounting for the low prevalence. Similarly, the occurrence of assemblage A in wolves and in some potential preys (deer) may be indicative of a different prey choice and consequent exposure to different G. duodenalis assemblages.

Our genotyping results, together with those reported in previous studies, indicate that assemblage A is predominant in wild mammals and that the genotype A1 is the most frequently found, followed by genotype A3. Studies in the early 80s, mainly based on the characterization of axenic strains of G. duodenalis, have indicated A1 as the most important zoonotic genotype within assemblage A (Thompson and Monis 2004). However, more recent studies have shown that genotype A2 is the predominant human type and that this genotype is rarely found in animals (Sprong et al. 2009). The present study has also confirmed the widespread distribution of genotype A3 among wild hoofed animals, including fallow deer, red deer, roe deer, moose, and wild boars (van der Giessen et al. 2006, Hamnes et al. 2007, Lalle et al. 2007, Sprong et al. 2009). This genotype belongs to subassemblage AIII (Cacciò et al. 2008), which has never been detected in humans or domestic animals, with the exception of a single cat isolate from Sweden (Lebbad et al. 2010), and is likely to have a minor zoonotic potential. It should also be noted that assemblage E has been very rarely identified in wild hoofed animals (Geurden et al. 2009, present study); the widespread occurrence of assemblage E in livestock suggests a recent adaption to hoofed animals, possibly following domestication.

In this survey, only two isolates from a wolf and a jackal were typed as assemblage B at the tpi locus, but this result was not confirmed by sequence analysis of the other loci, which supported the presence of assemblage A in these isolates. A lack of concordance in the assignment to specific assemblages was observed in four other isolates. This can be due to mixed infections (Lasek-Nesselquist et al. 2008), with preferential amplification of one assemblage over the other at a locus and the opposite at another locus, or to the occurrence of recombinants. Elucidation of this aspect, which is increasingly recognized as a major problem in Giardia genotyping studies (Sprong et al. 2009), requires the design of assemblage-specific primers and their application to single-cyst PCRs, which are technically demanding (Almeida et al. 2010).

The detection of G. microti in a wolf and a roe deer and of assemblage D in a red deer and 2 roe deer isolates is surprising, because G. microti infects rodents and assemblage D is believed to be specific for canids. It is difficult to distinguish between a mechanical passage of ingested cysts and an actual infection of the host. Certainly, wolves may ingest G. microti cysts by eating infected rodents, and cervids may be exposed by grazing on contaminated pastures; the low number of cysts in the feces suggests mechanical passage. However, the existence of a strong host specificity in Giardia can also be questioned. A recent study in Uganda (Johnston et al. 2010) reported evidence for cross-species transmission of multiple G. duodenalis assemblages among people, livestock, and primates. In particular, assemblage E, considered to be specific for hoofed animals, was detected in a red colobus. In a large-scale analysis of 978 humans and 1440 animal isolates of Giardia (Sprong et al. 2009), assemblages C to F were all found in noncanonical hosts, including humans, albeit at a very low frequency.

In summary, this survey has indicated a generally low prevalence of Giardia in wild mammals in Croatia, and genetic analysis has further shown that only a minority of isolates are potentially zoonotic. Although wildlife is unlikely to play an important role in the transmission of these parasites to humans, cycling between sylvatic and domestic animals is possible.

Footnotes

Acknowledgments

This work has been partially supported by a grant (FOOD-CT-2004-506122) from the 6th European Union Framework Program (Med-Vet-Net, a Network of Excellence for the Integrated Research on the Prevention and Control of Zoonoses). The authors thank Daniele Tonanzi for his excellent technical support.

Disclosure Statement

No competing financial interests exist.

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